CRISPR-P: A Web Tool for Synthetic Single-Guide RNA Design of CRISPR-System in Plants

Yang, Lei; Lü, Li; Liu, Haiyang; Li, Sen; Feng, Xing; Chen, Lingling

doi:10.1093/mp/ssu044

Cited by 631 publications

(419 citation statements)

References 12 publications

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“…The hda19-5 and hda19-6 alleles were generated from the same sgRNA. The sgRNAs used for mutagenesis in the generation of them were designed using the CRISPR-P program (Lei et al, 2014).…”

Section: Generation Of Mutant Arabidopsis Lines Using the Crispr/cas9mentioning

confidence: 99%

The Distinct Roles of Class I and II RPD3-Like Histone Deacetylases in Salinity Stress Response

et al. 2017

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ORCID IDs: 0000-0002-7750-6056 (M.U.); 0000-0002-3979-3947 (A.M.); 0000-0001-8288-0467 (M.S.).Histone acetylation is an essential process in the epigenetic regulation of diverse biological processes, including environmental stress responses in plants. Previously, our research group identified a histone deacetylase (HDAC) inhibitor (HDI) that confers salt tolerance in Arabidopsis (Arabidopsis thaliana). In this study, we demonstrate that class I HDAC (HDA19) and class II HDACs (HDA5/14/15/18) control responses to salt stress through different pathways. The screening of 12 different selective HDIs indicated that seven newly reported HDIs enhance salt tolerance. Genetic analysis, based on a pharmacological study, identified which HDACs function in salinity stress tolerance. In the wild-type Columbia-0 background, hda19 plants exhibit tolerance to high-salinity stress, while hda5/14/15/18 plants exhibit hypersensitivity to salt stress. Transcriptome analysis revealed that the effect of HDA19 deficiency on the response to salinity stress is distinct from that of HDA5/14/15/18 deficiencies. In hda19 plants, the expression levels of stress tolerance-related genes, late embryogenesis abundant proteins that prevent protein aggregation and positive regulators such as ABI5 and NAC019 in abscisic acid signaling, were induced strongly relative to the wild type. Neither of these elements was up-regulated in the hda5/14/15/18 plants. The mutagenesis of HDA19 by genome editing in the hda5/14/15/18 plants enhanced salt tolerance, suggesting that suppression of HDA19 masks the phenotype caused by the suppression of class II HDACs in the salinity stress response. Collectively, our results demonstrate that HDIs that inhibit class I HDACs allow the rescue of plants from salinity stress regardless of their selectivity, and they provide insight into the hierarchal regulation of environmental stress responses through HDAC isoforms.Histones are DNA-packaging proteins that provide stability to the genome by preventing physical genotoxicity (e.g. DNA breaks; Luger et al., 1997;Downs et al., 2007). They also have been considered to originally function as regulators of mRNA expression before the divergence of the Archaea and Eukarya (Ammar et al., 2012). A variety of chemical modifications (acetylation, methylation, phosphorylation, etc.) to the N tails of histones are one of the properties that enable the regulation of mRNA expression, which is generally conserved in eukaryotes (Jenuwein and Allis, 2001;Kouzarides, 2007). Chromatin possesses a diverse array of chemical moieties that allows it to contain and transmit information that is independent of the genetic code (i.e. epigenetic) and regulate gene expression levels. Epigenetic regulation is considered to be profoundly associated with plant development and adaptation to the environment. A complete understanding of the coordinated regulation of gene expression by histone modifications, however, is still lacking in plants. The role of epigenetic regulation in the abiotic stress response...

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“…The hda19-5 and hda19-6 alleles were generated from the same sgRNA. The sgRNAs used for mutagenesis in the generation of them were designed using the CRISPR-P program (Lei et al, 2014).…”

Section: Generation Of Mutant Arabidopsis Lines Using the Crispr/cas9mentioning

confidence: 99%

The Distinct Roles of Class I and II RPD3-Like Histone Deacetylases in Salinity Stress Response

et al. 2017

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“…A similar candidate target was suggested by three CRISPR designing tools (CRISPOR, CRISPR-P, and CRISPR-PLANT; Lei et al, 2014;Xie et al, 2014;Haeussler et al, 2016) and was selected for mutagenesis. A protospacer was designed using the ATTG_protospacer and AAAC_protospacer primers (Supplemental Table 3), and the annealed primers were cloned into pEn-Chimera according to Fauser et al (2014).…”

Section: Crispr Mutagenesismentioning

confidence: 99%

The Phosphate Fast-Responsive Genes PECP1 and PPsPase1 Affect Phosphocholine and Phosphoethanolamine Content

et al. 2018

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Phosphate starvation-mediated induction of the HAD-type phosphatases PPsPase1 (AT1G73010) and PECP1 (AT1G17710) has been reported in Arabidopsis (Arabidopsis thaliana). However, little is known about their in vivo function or impact on plant responses to nutrient deficiency. The preferences of PPsPase1 and PECP1 for different substrates have been studied in vitro but require confirmation in planta. Here, we examined the in vivo function of both enzymes using a reverse genetics approach. We demonstrated that PPsPase1 and PECP1 affect plant phosphocholine and phosphoethanolamine content, but not the pyrophosphate-related phenotypes. These observations suggest that the enzymes play a similar role in planta related to the recycling of polar heads from membrane lipids that is triggered during phosphate starvation. Altering the expression of the genes encoding these enzymes had no effect on lipid composition, possibly due to compensation by other lipid recycling pathways triggered during phosphate starvation. Furthermore, our results indicated that PPsPase1 and PECP1 do not influence phosphate homeostasis, since the inactivation of these genes had no effect on phosphate content or on the induction of molecular markers related to phosphate starvation. A combination of transcriptomics and imaging analyses revealed that PPsPase1 and PECP1 display a highly dynamic expression pattern that closely mirrors the phosphate status. This temporal dynamism, combined with the wide range of induction levels, broad expression, and lack of a direct effect on Pi content and regulation, makes PPsPase1 and PECP1 useful molecular markers of the phosphate starvation response.Plant growth is highly sensitive to a lack of phosphate (Pi); hence, the application of Pi fertilizers has become standard practice for high-throughput crop production (Cordell et al., 2009). Most phosphorus in the soil is not available to plants, as it is combined with other minerals or parts of organic compounds (Bieleski, 1973;Raghothama and Karthikeyan, 2005). Only a small fraction of soluble Pi (usually present at a concentration of ,10 mM in the soil) can be taken up by plants.Although growth is not optimal under limiting conditions, plants can withstand changing Pi concentrations within heterogeneous soils or in the external nutrient supply that can lead to reprogramming of their metabolism and architecture (Hammond et al., 2003;Péret et al., 2011;Plaxton and Tran, 2011). Root architecture can be modified to facilitate Pi uptake by favoring the development of lateral roots (at the expense of primary root elongation in many plants including Arabidopsis), increasing the density and length of root hairs, and limiting the development of aerial parts (López-Bucio et al., 2002;Svistoonoff et al., 2007;Gruber et al., 2013). Pi uptake mechanisms are enhanced at the root/soil interface, particularly through the stimulation of Pi transport activity (Mudge et al., 2002;Shin et al., 2004;Nussaume et al., 2011; Ayadi et al., 2015). In parallel, mobilization of Pi sources fr...

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“…Custom Perl scripts were used for probe annotation following the NetAffx procedures (Liu et al 2003). gRNAs were designed according to the method of Lei et al (2014) for 1000 randomly selected genes extracted from the P. trichocarpa or sPta717 genome. The resulting gRNA sequences were mapped to the respective genome using BatMis (Tennakoon et al 2012) to evaluate specificity according to Hsu et al (2013).…”

Section: Ngs Read Mappingmentioning

confidence: 99%

Exploiting genome variation to improve next-generation sequencing data analysis and genome editing efficiency in Populus tremula × alba 717-1B4

Xue

Alabady

Mohebbi

et al. 2015

Tree Genetics & Genomes

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Populus species are widely distributed across the Northern Hemisphere. The genetic diversity makes the genus an ideal study system for traits of ecological or agronomic significance. However, sequence variation between the genome-sequenced Populus trichocarpa Nisqually-1 and many other Populus species and hybrids poses significant challenges for research that employs sequence-sensitive approaches, such as next-generation sequencing and sitespecific genome editing. Using the routinely transformed genotype Populus tremula×alba 717-1B4 as a test case, we utilized established variant-calling pipelines with affordable re-sequencing (~20×) and publicly available transcriptome data to generate a variant-substituted custom genome (sPta717). The sPta717 genome harbors over 10 million SNPs or small indels relative to the P. trichocarpa v3 reference genome. When applied to RNA-Seq analysis, the fraction of uniquely mapped reads increased by 13-28 % relative to that obtained with the P. trichocarpa reference genome, depending on read length and sequence type. The enhanced mapping rates enabled detection of several hundred more expressed genes and improved the differential expression analysis. Similar improvements were observed for DNA-Seq and ChIP-Seq data mapping. The sPta717 genome is also instrumental in guide RNA (gRNA) design for CRISPR-mediated genome editing. We showed that a majority of gRNAs designed from the P. trichocarpa reference genome contain mismatches with the corresponding target sequences of sPta717, likely rendering those gRNAs ineffective in transgenic 717. A website is provided for querying the sPta717 genome by gene model or homology search. The same approach should be applicable to other outcrossing species with a closely related reference genome.

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CRISPR-P: A Web Tool for Synthetic Single-Guide RNA Design of CRISPR-System in Plants

Cited by 631 publications

References 12 publications

The Distinct Roles of Class I and II RPD3-Like Histone Deacetylases in Salinity Stress Response

The Distinct Roles of Class I and II RPD3-Like Histone Deacetylases in Salinity Stress Response

The Phosphate Fast-Responsive Genes PECP1 and PPsPase1 Affect Phosphocholine and Phosphoethanolamine Content

Exploiting genome variation to improve next-generation sequencing data analysis and genome editing efficiency in Populus tremula × alba 717-1B4

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